SITES OF METYRAPONE INHIBITION OF STEROID BIOSYNTHESIS BY RAT ADRENAL MITOCHONDRIA

1974 ◽  
Vol 76 (4) ◽  
pp. 703-711 ◽  
Author(s):  
Andres Carballeira ◽  
Su Chiau Cheng ◽  
Lawrence M. Fishman
Keyword(s):  
Inhibitory Effect ◽  
Side Chain ◽  
Steroid Biosynthesis ◽  
Side Chain Cleavage ◽  
Chain Cleavage ◽  
Low Concentrations ◽  
Wide Range ◽  
Generating System

ABSTRACT Rat adrenal mitochondrial preparations supplemented with an NADPH-generating system were incubated with various labelled substrates in order to evaluate further the action of metyrapone on the utilization of cholesterol for steroid biosynthesis1). The formation of pregnenolone from [4-14C] cholesterol (0.5, 1.0 and 2.0 μCi) in 5, 10 and 15 min incubations was decreased by 72–82 % in the presence of metyrapone (0.5 mm). Similarly, the generation of labelled side chain fragments from [26-14C]-cholesterol was depressed 36–42 % by 0.2 mm metyrapone and 65–70 % by 1.0 mm inhibitor during 30, 60 and 90 min incubations. Metyrapone inhibition of the side chain cleavage was not observed, however, if cholesterol was replaced as substrate by its C-20 hydroxylated analog: The formation of pregnenolone from [7-3H]20α-hydroxycholesterol (0.5, 1.0 and 2.0 μCi), also an NADPH-mediated mitochondrial reaction, was not affected by similar concentrations of metyrapone, indicating that the inhibition observed with cholesterol as substrate is not related to non-specific toxic effects, to interference with NADPH generation or to impairment of NADPH function in the mitochondrial electron transport system. Parallel incubations with [4-14C] 11-deoxycorticosterone and with [4-14C] cholesterol over a wide range of inhibitor concentrations (0.01–1.0 mm) demonstrated that the effects of metyrapone on 11β-hydroxylation and on the side chain cleavage were dose-related; at low concentrations, however, metyrapone was a more potent inhibitor of 11β-hydroxylation than of cholesterol conversion to pregnenolone. These studies demonstrate clearly in the rat adrenal the dual inhibitory effect of metyrapone sug-gested by previous in vivo and in vitro observations in man.

Alteration in the expression of genes for cholesterol side-chain cleavage enzyme and 21-hydroxylase by hypophysectomy and ACTH administration in the rat adrenal

1990 ◽  
Vol 4 (3) ◽  
pp. 239-245 ◽  
Author(s):  
T. Imai ◽  
H. Seo ◽  
Y. Murata ◽  
M. Ohno ◽  
Y. Satoh ◽  
...  
Keyword(s):  
Steady State ◽  
Adrenal Weight ◽  
Side Chain ◽  
Total Rna ◽  
Side Chain Cleavage ◽  
Chain Cleavage ◽  
Acth Treatment ◽  
Steady State Levels ◽  
Cytochrome P 450

ABSTRACT The changes in steady-state levels of mRNA for cholesterol side-chain cleavage cytochrome P-450 (P-450scc) and steroid 21-hydroxylase cytochrome P-450 (P-450c21) caused by hypophysectomy and ACTH treatment were determined in rat adrenals. Hypophysectomy caused marked decreases in adrenal weight and total RNA per gland. Administration of ACTH resulted in increases in adrenal weight and total RNA. A significant correlation between the amount of RNA and adrenal weight was observed. Both P-450scc and P-450c21 mRNAs were decreased by hypophysectomy and increased by ACTH treatment. P-450scc mRNA decreased to 20% and P-450c21 mRNA to 76% of control values 1 day after hypophysectomy. ACTH caused a significant increase in P-450scc mRNA after 3 h. However, a significant increase in P-450c21 mRNA was observed 12 h after administration of ACTH. These results are concordant with previous studies in vitro utilizing cultured adrenocortical cells. Moreover, the induction of steady-state levels of P-450scc mRNA was faster than that observed by other investigators in studies in vitro. These results may indicate that integrity of the adrenal gland in vivo is important for the action of ACTH.

Effects of polymethoxylated flavone metabolites on apoB100 secretion and MTP activity in Huh7.5 cells

2021 ◽  
Vol 18 ◽  
Author(s):  
Danielle R. Gonçalves ◽  
Thais B. Cesar ◽  
John A. Manthey ◽  
Paulo I. Costa
Keyword(s):  
Lipid Synthesis ◽  
Dependent Manner ◽  
Transfer Protein ◽  
Low Concentrations ◽  
Wide Range ◽  
Cell Assays ◽  
Polymethoxylated Flavones ◽  
High Concentrations

Background: Citrus polymethoxylated flavones (PMFs) reduce the synthesis of liver lipoproteins in animal and in vitro cell assays, but few studies have evaluated the direct effects of their metabolites on this highly regulated process. Objective: To investigate the effects of representative metabolites of PMF on the secretion of liver lipoproteins using the mammalian cell Huh7.5. Method: In this study, the influences of three PMFs and five previously isolated PMF metabolites on hepatic apoB-100 secretion and microsomal transfer protein (MTP) activity were evaluated. Tangeretin (TAN), nobiletin (NOB) and 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF), and their glucuronides (TAN-Gluc, NOB-Gluc and HMF-Gluc) and oxidatively demethylated metabolites (TAN-OH, NOB-OH, HMF-OH) were incubated with Huh7.5 cells to measure their inhibitory effects on lipid synthesis. Results: The results showed that TAN, HMF and TAN-OH reduced the secretion of apoB-100 in a dose-dependent manner, while NOB and the other tested metabolites showed no inhibition. MTP activity in the Huh7.5 cells was significantly reduced in the presence of low concentrations of TAN, and in high concentrations of NOB-OH. This study also showed that PMFs and PMF metabolites produced a wide range of effects on apoB-100 secretion and MTP activity. Conclusion: The results suggest that while PMFs and their metabolites control dyslipidemia in vivo, the inhibition of MTP activity cannot be the only pathway influenced by these compounds.

STEROID BIOSYNTHESIS IN VITRO BY A VIRILIZING SUPRARENAL TUMOUR

1963 ◽  
Vol 44 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Roversi G. D. ◽  
Polvani F. ◽  
Bompiani A. ◽  
Neher R.
Keyword(s):  
Adrenal Adenoma ◽  
Tumour Tissue ◽  
Side Chain ◽  
Steroid Biosynthesis ◽  
Before And After

ABSTRACT A case of virilizing adrenal adenoma is described. The tumour was incubated with progesterone-4-14C. In the extract the following steroids were identified chromatographically, in order of decreasing quantity and radioactivity: 17α-hydroxyprogesterone, androst-4-ene-3,17-dione, corticosterone (11β,21 -dihydroxy-pregn-4-ene-3,20-dione), cortisol (11β,17,21-trihydroxy-pregn-4-ene-3,20-dione) and 11-deoxycortisol. This indicates either an increase in 17α-hydroxylation and side chain split, or a partial blockage of 21-hydroxylation, or a combination of both in the tumour tissue. The absence of the 3β-hydroxy-dehydrogenase demonstrated histochemically in the tumour and the examination of the urinary 17-ketosteroids before and after removal of the neoplasm, suggested the same abnormal biosynthetic pattern in vivo with regard to the level of the endogenous Δ5-precursors.

scholarly journals Studies on Analogues of L-Cysteine and L-Cystine

Blood ◽  
1956 ◽  
Vol 11 (1) ◽  
pp. 1-10 ◽  
Author(s):  
AUSTIN S. WEISBERGER ◽  
LEIF G. SUHRLAND ◽  
JOSEPH SEIFTER
Keyword(s):  
Amino Acids ◽  
Spatial Configuration ◽  
Spatial Relationship ◽  
Inhibitory Effect ◽  
Selenium Compounds ◽  
Low Concentrations ◽  
Relationship Of ◽  
Normal Cellular Function

Abstract The amino acids L-cysteine and L-cystine appear to have an important role in the metabolism of leukocytes. Decreased availability of these amino acids may therefore have important effects on leukocytes. The possibility of decreasing the influx of radioactive L-cystine into leukemic leukocytes was investigated by exposing the leukocytes to various analogues of cysteine (cystine) prior to incubation with S35 L-cystine. It was found that a highly specific structural and spatial configuration is required to decrease the influx of S35 L-cystine. Thus unlabeled L-cysteine is effective in decreasing the incorporation of radioactive L-cystine. However, analogues of cystine in which there is modification or substitution of the sulfhydryl, amino or carboxyl group do not decrease the influx of S35 L-cystine. Furthermore, any alteration in the spatial relationship of the sulfhydryl and amino groups of L-cysteine also results in a loss of the ability of an analogue to decrease the incorporation of S35 L-cystine. Of the compounds studied and in the concentrations employed, only unlabeled L-cysteine, selenium cystine and phenyl selenium cysteine were effective. Selenium cystine is identical with cystine except that selenium replaces the sulfur in the molecule. Phenyl selenium cysteine is also closely related structurally to cysteine. The mechanism of action of selenium cystine and phenyl selenium cysteine in decreasing the influx of S35 L-cystine is not known. Other selenium compounds tested were ineffective. These compounds may exert their inhibitory effect by (a) competitive combination with specific intracellular receptors for L-cysteine (L-cystine), (b) inactivation of enzymes or compounds essential for normal cellular function, (c) alteration in membrane permeability or (d) a toxic effect of selenium. Since selenium cystine and phenyl selenium cystine are inhibitory in low concentrations in vitro, these compounds may have important effects on leukemic leukocytes in vivo.

scholarly journals The Xenobiotic Compound 1,4-Bis[2-(3,5-Dichloropyridyloxy)]Benzene Is an Agonist Ligand for the Nuclear Receptor CAR

2000 ◽  
Vol 20 (9) ◽  
pp. 2951-2958 ◽  
Author(s):  
Iphigenia Tzameli ◽  
Pavlos Pissios ◽  
Erin G. Schuetz ◽  
David D. Moore
Keyword(s):  
Ligand Binding ◽  
Nuclear Receptor ◽  
Metabolic Response ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Inverse Agonists ◽  
Wide Range ◽  
Xenobiotic Compound

ABSTRACT A wide range of xenobiotic compounds are metabolized by cytochrome P450 (CYP) enzymes, and the genes that encode these enzymes are often induced in the presence of such compounds. Here, we show that the nuclear receptor CAR can recognize response elements present in the promoters of xenobiotic-responsive CYP genes, as well as other novel sites. CAR has previously been shown to be an apparently constitutive transactivator, and this constitutive activity is inhibited by androstanes acting as inverse agonists. As expected, the ability of CAR to transactivate the CYP promoter elements is blocked by the inhibitory inverse agonists. However, CAR transactivation is increased in the presence of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), the most potent known member of the phenobarbital-like class of CYP-inducing agents. Three independent lines of evidence demonstrate that TCPOBOP is an agonist ligand for CAR. The first is that TCPOBOP acts in a dose-dependent manner as a direct agonist to compete with the inhibitory effect of the inverse agonists. The second is that TCPOBOP acts directly to stimulate coactivator interaction with the CAR ligand binding domain, both in vitro and in vivo. The third is that mutations designed to block ligand binding block not only the inhibitory effect of the androstanes but also the stimulatory effect of TCPOBOP. Importantly, these mutations do not block the apparently constitutive transactivation by CAR, suggesting that this activity is truly ligand independent. Both its ability to target CYP genes and its activation by TCPOBOP demonstrate that CAR is a novel xenobiotic receptor that may contribute to the metabolic response to such compounds.

scholarly journals Insulin and IGF-I are necessary for FSH-induced cytochrome P450 aromatase but not cytochrome P450 side-chain cleavage gene expression in oestrogenic bovine granulosa cells in vitro

2002 ◽  
Vol 174 (3) ◽  
pp. 499-507 ◽  
Author(s):  
JM Silva ◽  
CA Price
Keyword(s):  
Gene Expression ◽  
Cytochrome P450 ◽  
Granulosa Cells ◽  
Side Chain ◽  
Mrna Levels ◽  
Side Chain Cleavage ◽  
P450 Aromatase ◽  
Chain Cleavage ◽  
Igf I

The earliest biochemical indicators of ovarian follicle deviation in cattle include lower oestradiol and free IGF concentrations in subordinate compared with dominant follicles. We determined if decreases in FSH, IGF-I or insulin cause decreased P450 aromatase (P450arom) or P450 cholesterol side-chain cleavage (P450scc) mRNA expression in oestrogenic bovine granulosa cells in vitro. In the first experiment, cells obtained from small follicles (2-5 mm diameter) were cultured in serum-free medium supplemented with physiological concentrations of FSH, IGF-I and insulin for 4 days. A decrease in specific hormone concentration was produced by replacing 70% of spent medium with medium devoid of FSH, insulin, or insulin and IGF-I on day 4 and again on day 5 of culture. Cultures were terminated on day 7. A reduction in FSH concentrations during the last 3 days of culture decreased P450arom and P450scc mRNA levels. A reduction in insulin reduced P450arom but not P450scc mRNA levels, and a reduction of both insulin and IGF-I concentrations further decreased P450arom mRNA levels and decreased P450scc mRNA levels. In a second experiment, cells obtained from small follicles (2-5 mm diameter) were cultured with insulin (100 ng/ml) without FSH for 4 days, and then insulin was withdrawn from the culture and FSH added for a further 3 days. The withdrawal of insulin decreased (P<0.02) oestradiol accumulation and reduced P450arom mRNA to below detectable levels, but did not affect P450scc mRNA levels. The addition of FSH transiently increased oestradiol secretion and P450arom mRNA levels, but P450arom mRNA levels were undetectable at the end of the culture period. The addition of FSH significantly enhanced P450scc mRNA levels and progesterone accumulation. These data demonstrated that a reduction of insulin-like activity reduced aromatase gene expression in bovine follicles without necessarily affecting progesterone synthetic capability, and thus may initiate follicle regression in cattle at the time of follicle divergence.

scholarly journals Evaluation of relative roles of LH and FSH in regulation of differentiation of Leydig cells using an ethane 1,2-dimethylsulfonate-treated adult rat model

2003 ◽  
Vol 176 (1) ◽  
pp. 151-161 ◽  
Author(s):  
V Sriraman ◽  
MR Sairam ◽  
AJ Rao
Keyword(s):  
Leydig Cells ◽  
Serum Testosterone ◽  
Side Chain ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Lh Receptor ◽  
Side Chain Cleavage ◽  
Chain Cleavage ◽  
Cleavage Enzyme

The relative role of LH and FSH in regulation of differentiation of Leydig cells was assessed using an ethane 1,2-dimethylsulfonate (EDS)-treated rat model in which endogenous LH or FSH was neutralized from day 3 to day 22 following EDS treatment. Serum testosterone and the in vitro response of the purified Leydig cells to human chorionic gonadotropin (hCG) was monitored. In addition RNA was isolated from the Leydig cells to monitor the steady-state mRNA levels by RT-PCR for 17alpha-hydroxylase, side chain cleavage enzyme, steroidogenic acute regulatory protein (StAR), LH receptor, estrogen receptor (ER-alpha) and cyclophilin (internal control). Serum testosterone was undetected and the isolated Leydig cells secreted negligible amount of testosterone on stimulation with hCG in the group of rats that were treated with LH antiserum following EDS treatment. RT-PCR analysis revealed the absence of message for cholesterol side chain cleavage enzyme and 17alpha-hydroxylase although ER-alpha and LH receptor mRNA could be detected, indicating the presence of undifferentiated precursor Leydig cells. In contrast, the effects following deprival of endogenous FSH were not as drastic as seen following LH neutralization. Deprival of endogenous FSH in EDS-treated rats led to a significant decrease in serum testosterone and in vitro response to hCG by the Leydig cells. Also, there was a significant decrease in the steady-state mRNA levels of 17alpha-hydroxylase, cholesterol side chain cleavage enzyme, LH receptor and StAR as assessed by a semiquantitative RT-PCR. These results establish that while LH is obligatory for the functional differentiation of Leydig cells, repopulation of precursor Leydig cells is independent of LH, and also unequivocally establish an important role for FSH in regulation of Leydig cell function.

scholarly journals Androgen profiles during pubertal Leydig cell development in mice

Reproduction ◽  
2010 ◽  
Vol 140 (1) ◽  
pp. 113-121 ◽  
Author(s):  
Xiufeng Wu ◽  
Ramamani Arumugam ◽  
Ningning Zhang ◽  
Mary M Lee
Keyword(s):  
Leydig Cell ◽  
Developmental Changes ◽  
Side Chain ◽  
Adult Rat ◽  
Testosterone Production ◽  
Chain Cleavage ◽  
Cleavage Enzyme ◽  
Species Specific

Postnatal Leydig cell (LC) development in mice has been assumed empirically to resemble that of rats, which have characteristic hormonal profiles at well-defined maturational stages. To characterize the changes in LC function and gene expression in mice, we examined reproductive hormone expression from birth to 180 days, and quantified in vivo and in vitro production of androgens during sexual maturation. Although the overall plasma androgen and LH profiles from birth through puberty were comparable to that of rats, the timing of developmental changes in androgen production and steroidogenic capacity of isolated LCs differed. In mice, onset of androgen biosynthetic capacity, distinguished by an acute rise in androstenedione and testosterone production and an increased expression of the steroidogenic enzymes, cytochrome P450 cholesterol side-chain cleavage enzyme and 17α-hydroxylase, occurred at day 24 (d24) rather than at d21 as reported in rats. Moreover, in contrast to persistently high testosterone production by pubertal and adult rat LCs, testosterone production was maximal at d45 in mice, and then declined in mature LCs. The murine LCs also respond more robustly to LH stimulation, with a greater increment in LH-stimulated testosterone production. Collectively, these data suggest that the mouse LC lineage has a delayed onset, and that it has an accelerated pace of maturation compared with the rat LC lineage. Across comparable maturational stages, LCs exhibit species-specific developmental changes in enzyme expression and capacity for androgen production. Our results demonstrate distinct differences in LC differentiation between mice and rats, and provide informative data for assessing reproductive phenotypes of recombinant mouse models.

A Comparative Study of the Effects of Adrenoceptor Antagonists on Platelet Aggregation and Thromboxane Generation

1985 ◽  
Vol 54 (02) ◽  
pp. 480-484 ◽  
Author(s):  
I A Greer ◽  
J J Walker ◽  
M McLaren ◽  
A A Calder ◽  
C D Forbes
Keyword(s):  
Platelet Aggregation ◽  
Vascular Disease ◽  
Platelet Rich Plasma ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Wide Range ◽  
The Right

SummaryPlatelet aggregation and thromboxane A2 have been implicated in the pathogenesis of several forms of vascular disease. The aim of this study was to determine the effect of a wide range of adrenoceptor antagonists on platelet aggregation, and thromboxane A2 production, from normal human platelet rich plasma in vitro. Labetalol, pindolol and propranolol inhibited platelet aggregation to collagen in a dose dependent manner. Increasing the concentration of collagen “shifted” the dose response curve to the right. These 3 drugs also significantly inhibited thromboxane A2 generation in response to collagen but not to arachidonic acid. This effect was independent of any inhibitory effect of these drugs on platelet aggregation, and occurred at a drug concentration close to that obtained in vivo. Atenolol, metoprolol, prazosin and timolol were similarly assessed but had no effect on either platelet aggregation or thromboxane A2 generation. This ability of labetalol, pindolol, and propranolol to inhibit platelet aggregation and thromboxane generation, may be of clinical benefit in view of the increasing evidence implicating thromboxane A2 in the pathogenesis of vascular disease.

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